|
| Status |
Public on Jan 08, 2013 |
| Title |
S-phase 118077B Top S6 agi |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
EBV-transformed lymphoblast cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: EBV-transformed lymphoblast cell line
gender: male
cell cycle phase: S-phase
treatment: male carrying 750kb amplification on the short arm of chromosome 4
|
| Treatment protocol |
Cells were stained with DyeCycle Orange (Invitrogen) according to the manufacturer’s protocol. In short, cells were washed with 1xPBS and incubated at a concentration of 10^6 cells/ml at 37°C for 30 minutes after adding DyeCycle orange at 2µl/10^6 cells. Cells of lymphoblastoid cell lines at ~5*10^6cells/ml were sorted using a FACS Vantage SE or a FACSAria III with FACS DiVa software (BD Biosciences).
|
| Growth protocol |
Epstein-Barr virus (EBV) immortalized lymphoid cells were cultured in DMEM/F12 medium (Gibco) with 10% fetal bovine Serum (Thermo Scientific) at 37°C at 5%CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genome of collected single cells was amplified using the Sureplex amplification system (BlueGnome).
|
| Label |
Cy5
|
| Label protocol |
150ng of test-samples and reference were labeled for 2h by random primer labeling (BioPrime aCGH Genomic Labeling System; Invitrogen) using respectively Cy5- and Cy3-dCTPs (GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
SureRef male reference DNA (BlueGnome
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Kreatech male reference DNA
gender: male
|
| Treatment protocol |
Cells were stained with DyeCycle Orange (Invitrogen) according to the manufacturer’s protocol. In short, cells were washed with 1xPBS and incubated at a concentration of 10^6 cells/ml at 37°C for 30 minutes after adding DyeCycle orange at 2µl/10^6 cells. Cells of lymphoblastoid cell lines at ~5*10^6cells/ml were sorted using a FACS Vantage SE or a FACSAria III with FACS DiVa software (BD Biosciences).
|
| Growth protocol |
Epstein-Barr virus (EBV) immortalized lymphoid cells were cultured in DMEM/F12 medium (Gibco) with 10% fetal bovine Serum (Thermo Scientific) at 37°C at 5%CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genome of collected single cells was amplified using the Sureplex amplification system (BlueGnome).
|
| Label |
Cy3
|
| Label protocol |
150ng of test-samples and reference were labeled for 2h by random primer labeling (BioPrime aCGH Genomic Labeling System; Invitrogen) using respectively Cy5- and Cy3-dCTPs (GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
Hybridization and washing of 24sure arrays followed the protocol provided by the manufacturer.
|
| Scan protocol |
Scanning was performed using a DNA-microarray scanner (Agilent Technologies)
|
| Description |
whole genome amplified singl-cell DNA
|
| Data processing |
GenePix Pro 6.0 software (Axon - Molecular Devices) was used for feature extraction and to normalize the data so the ratio of means for all features equals 1. All further analyses were performed in R (version 2.13.2 – www.r-project.com). Log2 intensity ratios with a signal/noise ratio of less than 2 were excluded from the analyses. The remaining intensities were made consistent within each array using median correction as implemented in R using the limma package. Next, the log2 intensity ratios of replicate probes were averaged using the R-package snapCGH. Technical GC-bias correction was performed by subtracting from each probe’s log2 intensity ratio the corresponding value from a mean locally weighted regression between the log2 intensity ratios and the %GC-content of the corresponding micro-array probe locus for all G- and G2/M-phase samples per batch. Using the spline function in R missing data in this mean lowess regression curve were filled in before it was used for the correction of log2 intensity ratios.
|
| |
|
| Submission date |
Jun 15, 2012 |
| Last update date |
Jan 08, 2013 |
| Contact name |
Niels Van der Aa |
| Organization name |
KU Leuven
|
| Street address |
Herestraat 49 bus 602
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL15703 |
| Series (2) |
| GSE38758 |
Identification of DNA-replication domains in single S-phase cells: part 1 whole genome amplified single-cell DNA |
| GSE38761 |
Identification of DNA-replication domains in single S-phase cells |
|
